Signal Molecules as Cell Penetration Agents

ABSTRACT

Novel cell penetrating agents for intracellular delivery of desired cargo, including proteins. Use of cell penetrating agents to deliver cargos to the interior of cells and cellular compartments and organelles is transformative for diagnostic, therapeutic, and research processes.

This application claims priority to U.S. Ser. No. 62/290,629 filed Feb.3, 2016, which is expressly incorporated by reference herein it isentirety.

Agents such as peptides have been discovered or designed to be rapidlyinternalized by eukaryotic cells. Such cell-penetrating agents, whichincludes but are not limited to peptides, mediate penetration of theplasma membrane, permitting delivery of one or more macromolecules, alsotermed a cargo or cargos, to the cell interior. A cargo may be aprotein, nucleic acid, liposome, drug, etc.

Typical cell penetrating peptides (CPP) are 10 to 30 amino acids inlength. Typical CPP are classified as one of arginine-rich CPP,amphipathic and lysine-rich CPP, or hydrophobic CPP. When themacromolecule is a protein, CPP have been attached to the N-terminus,the C-terminus, and intermediate positions, using a variety of covalentor non-specific hydrophobic linkage methods, e.g., using cysteine thiolsfor targeting.

Representative known CPP include the following:

Lysine rich CPPs and others derived from translocation domains PeptideOrigin Sequence Cargo types Tat HIV-Tat protein PGRKKRRQRRPPQProtein/peptide/siRNA/ SEQ ID NO: 6 (46-58) liposome/nanoparticlePenetratin Homeodomain RQIKIWFQNRRMKWKK peptide/siRNA/ SEQ ID NO: 7liposome Transportan Galanin-mastoparan GWTLNSAGYLLGKINLKALAALAKKILProtein/peptide/siRNA SEQ ID NO: 8 Dat Dopamine FREKLAYIAPProtein/peptide/siRNA SEQ ID NO: 9 transporter VP-22 HSV-1 structuralDAATATRGRSAASRPTERPRAPAR- Protein protein SASRPRRPVD SEQ ID NO: 10Amphipathic peptides Peptide Origin Sequence Cargo types MPGHIV Gp41-SV40 GALFLGFLGAAGSTMGAWSQPKKKRKV siRNA/ODN/plasmidSEQ ID NO: 11 Pep-1 Trp-rich motif- KETWWETVWVTEWSQPKKKRKVProtein/peptide SV40 NLS SEQ ID NO: 12 MAP Chimeric KALAKALAKALASmall molecule/ SEQ ID NO: 13 plasmid SAP Proline-rich motifVRLPPPVRLPPPVRLPPP protein/peptide SEQ ID NO: 14 PPTG1 ChimericGLFRALLRLLRSLWRLLLRA Plasmid SEQ ID NO: 15Arginine rich and other cell-penetrating peptides Peptide OriginSequence Cargo types Oligoarginine Chimeric Agr8 or Arg9 siRNA/ODNProtein/peptide/ SEQ ID NO: 16 and 17 hCT (9-32) HumanLGTYTQDFNKTFPQTAIGVGAP Protein/plasmid DNA SEQ ID NO: 18 calcitonin SynBProtegrin RGGRLSYSRRRFSTSTGR Doxorubicin SEQ ID NO: 19 Pvec Murine VE-LLIILRRRIRKQAHAHSK Protein/peptide SEQ ID NO: 20 cadherin

CPP are promising for use as diagnostics and therapeutics. Whilecurrently used in clinical trials and as research tools, limitationssuch as their penetration efficiency and endosomal entrapment haveprevented their broad adoption as a method to alter the intracellularenvironment. Transcription-transactivating (TAT) peptides are shortsignal sequences that mediate transport of proteins across the membranesof many cells. TAT peptides were believed to directly mediate transportacross phospholipid bilayers; they can drive uptake of large proteinsthat could not cross the membrane without an active uptake process. TATpeptides attach to membrane receptors and cause internalization incoated pits. Constructs are known that can be internalized by processesthat rely on recognition of short TAT peptides attached as C or Nterminal fusions. The growing consensus is that most, if not all, CPPare rapidly internalized by receptor-mediated endocytosis. However, theyare not efficiently released into the cytoplasm, likely due to highaffinity interactions between CPP and their receptors. Thus, covalentlyor hydrophobically linked CPP-cargos remain trapped in the endosomes.These cargo proteins also required purification as CPP adducts. Thisresulted in eukaryote expression complicated by binding to importmachinery via the CPP, and complicated handling of the CPP because manydesirable products were rendered potentially hazardous by the CPP tag.

The inventive system and method overcomes these and other detriments.The inventive system and method extends utility of TAT peptide andrelated CPP constructs by expressing CPP fusions of small proteins thatstrongly bind other proteins.

SUMMARY

In one aspect, a composition for intracellular delivery of a biomoleculeis provided, where the composition comprises a cell penetrating peptide(CPP) or cell penetrating agent (CPA) covalently linked to an adapter,and a cargo molecule covalently linked to an adapter binding molecule,and the composition formed by non-covalent linkage between the adapterand adapter binding molecule. The composition comprises a cellpenetrating peptide (CPP) or cell penetrating agent (CPA) covalentlylinked to an adapter, the adapter non-covalently linked to an adapterbinding molecule, and a cargo molecule covalently linked to the adapterbinding molecule, where the composition provides for intracellulardelivery of the cargo. The composition for intracellular delivery of abiomolecule is provided, the composition comprises a cell penetratingpeptide (CPP) or cell penetrating agent (CPA) covalently linked to anadapter, the adapter non-covalently linked to an adapter bindingmolecule, and the adapter binding molecule covalently linked to a cargo.

In one embodiment, the adapter is calmodulin or a calcium bindingprotein and the adapter binding molecule is a calmodulin bindingpeptide. In one embodiment, the CPP is selected from the groupconsisting of Tat, penetratin, transportan, Dat, VP-22, amphipathicpeptides, MPG, Pep-1, MAP, SAP, PPTG1, arginine rich peptides,oligoarginine, hCT (9-32), SynB, and Pvec. In one embodiment, the CPA isselected from a receptor or transporter ligand, and the receptor isselected from the group consisting of insulin receptor, beta2-adrenergic receptor, folate receptor, the N-methyl-D-aspartic acid(NMDA) receptor, opiate receptors, cannabinoid receptor, andcombinations thereof, and the transporter is selected from the groupconsisting of dopamine transporter, serotonin transporter,norepinephrine transporter, endothelial glucose transporter, andcombinations thereof.

In one embodiment, the adapter binding molecule is further attached to asequence that localizes the cargo to a cellular location or organellethat is selected from the group consisting of nucleus, peroxisome,mitochondria, endoplasmic reticulum, Gogli, and combinations thereof. Inone embodiment, the sequence is a nuclear localization sequence.

In various embodiments, the cargo is selected from the group consistingof a protein, a drug, a liposome, a nucleic acid, and combinationsthereof.

In various embodiments, the non-covalent linkage between the adapter andthe adapter binding molecule is reversible. In one embodiment, at leastone characteristic of an intracellular environment promotes reversal ofthe non-covalent linkage between the adapter and the adapter bindingmolecule, resulting in release of the cargo from the CPP. In oneembodiment, the characteristic is an intracellular calciumconcentration.

In one embodiment, the cargo is selected from the group consisting of amodulator of transcription in the cell, a probe that measures a propertyof the cell interior, and an enzyme. In one embodiment, the enzyme is akinase or a phosphatase. In one embodiment, the enzyme is modified to beconstitutively active. In one embodiment, the probe is an oxidationmonitor, a nitric oxide (NO) sensor, or a pH sensor. In one embodiment,the cargo is a nucleic acid and the adapter binding molecule iscovalently linked to a nucleic acid binding protein.

In another aspect, a method for delivering a cargo inside a cell isprovided. In one embodiment, the method comprises forming a complex bycontacting a cell penetrating peptide (CPP) or cell penetrating agent(CPA) covalently linked to an adapter, with a cargo molecule covalentlylinked to an adapter binding molecule, under conditions suitable forforming a non-covalent bond between the adapter and adapter bindingmolecule, and contacting the cell with the complex under conditionssufficient to result in delivery of the cargo inside the cell. In oneembodiment, the cell penetrating peptide (CPP) or cell penetrating agent(CPA) covalently linked to an adapter and the cargo molecule covalentlylinked to an adapter binding molecule are added separately to culturemedia containing the cell, and the complex forms in the culture media.

In one embodiment, the adapter is calmodulin or a calcium bindingprotein and the adapter binding molecule is a calmodulin bindingpeptide. In one embodiment, the CPP is selected from the groupconsisting of Tat, Penetratin, Transportan, Dat, VP-22, Amphipathicpeptides, MPG, Pep-1, MAP, SAP, PPTG1, arginine rich peptides,Oligoarginine, hCT (9-32), SynB, and Pvec. In one embodiment, the CPA isselected from the group consisting of a receptor or transporter ligand.In one embodiment, the receptor is selected from the group consisting ofinsulin receptor, beta 2-adrenergic receptor, folate receptor, theN-methyl-D-aspartic acid (NMDA) receptor, opiate receptors, andcannabinoid receptor, and the transporter is selected from the groupconsisting of dopamine transporter, serotonin transporter,norepinephrine transporter, and endothelial glucose transporter.

In one embodiment, the adapter binding molecule is also bound to alocalization sequence. In one embodiment, the localization sequence is anuclear localization sequence.

In one embodiment, the cargo is at least one of a protein, a drug, aliposome, or a nucleic acid.

In one embodiment, the non-covalent linkage between the adapter and theadapter binding molecule is reversible. In various embodiments, at leastone characteristic of an intracellular environment promotes reversal ofthe non-covalent linkage between the adapter and the adapter bindingmolecule, resulting in release of the cargo from the CPP. In oneembodiment, the characteristic is an intracellular calciumconcentration.

In one embodiment, the cargo is selected from the group consisting of amodulator of transcription in the cell, a probe that measures a propertyof the cell interior, an enzyme, and combinations thereof. In oneembodiment, the enzyme is a kinase or a phosphatase. In one embodiment,the enzyme is modified to be constitutively active. In one embodiment,the probe is selected from the group consisting of an oxidation monitor,a NO sensor, a pH sensor, and combinations thereof.

In one embodiment, the cargo is a nucleic acid and the adapter bindingmolecule is covalently linked to a nucleic acid binding protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a scheme for cellular uptake of cargo tagged with a cellpenetrating peptide (CPP), where uptake of CPP bound cargo proceedsclockwise from upper left by binding to membrane and invagination;depending on the CPP tag, cargo may be targeted to internal compartmentssuch as nuclei, mitochondria, etc., or to the cytoplasm.

FIG. 2 shows one scheme for the adapter mediated cellular uptake ofcargo; association can be mediated by protein-protein or protein-ligandinteractions. Clockwise from upper left, association mediated byprotein-protein or protein-ligand interactions, binding to cellmembrane, internalization, and redistribution to internal compartments.

FIGS. 3A and 3B show the basic fluorescence resonance energy transfer(FRET) experiment to detect protein-protein interactions. FIG. 3A showsthat excitation of D leads to emission from A only when A and D are inproximity due to complex formation, also reducing donor emission. FIG.3B shows that when A and D are not in proximity, there is no emissionfrom A. Examples of donor acceptor pairs with good overlap include AlexaFluor 488 and Alexa Fluor 647.

FIGS. 4A and 4B show ribbon representations of the three dimensionalstructure of calmodulin (CaM), used here as an adapter. Structures ofCa²⁺-CaM bound to a canonical target peptide in the center of themolecule (FIG. 4A) and in the dumbbell-shaped conformation in theabsence of target (FIG. 4B). The central helix breaks during recognitionand binding, allowing calmodulin to wrap around the target. Ca²⁺ areshown as small spheres; the protein is less ordered in the absence ofCa²⁺ (not shown). Free N and C termini are visible.

FIG. 5 shows sequence alignment of human calmodulin 1 (CALM) and four“calmdulin-like proteins”. The similarity of these human calmodulinhomologs is much less than the similarity of human and C. eleganscalmodulin; less than 2% of the positions are identically conserved. Thesequences shown are, from top to bottom, SEQ ID NOS: 21-25.

FIG. 6 shows the amino acid sequence of TAT-CaM, a CPP taggedcalmodulin. The short CPP binding sequence (SEQ ID NO: 26), whichcontains amino acids 47-57 of SEQ ID NO: 6 (TAT), is located directlybefore the NOT1 site, which is followed by CaM (SEQ ID NO: 21).

FIGS. 7A and 7B show confocal microscopy images demonstrating uptake ofa fluorescence labeled enzyme, neuronal nitric oxide synthase (nNOS),mediated by a CPP linked calmodulin adapter. Projection confocal imageof labeled nNOS three hours after onset of TAT-CaM mediated uptake byBHO cells. The indicated nucleus is stained blue; labeled nNOS isstained yellow with DyLight 540. FIG. 7A shows nNOS added without CPPadapter. Background still shows stained nNOS after washing with media.Some nNOS adheres to the cell surface; three-dimensional cross sectionsshow no nNOS inside cells. FIG. 7B shows that in the presence ofTAT-CaM, a large amount of nNOS is rapidly and actively pumped insidethe cell, clearing the intracellular space and protecting nNOS fromremoval by washing. The cell boundary is now visible because cytoplasmis stained by released nNOS. three dimensional cross sections confirmthat labeled nNOS is inside the cells. Circles (yellow) inside cells arelabeled endosomes.

FIGS. 8A-8F show a design of TAT-CaM and cargo proteins, according toone embodiment, and dissociation data for various embodiments. FIG. 8Ashows a schematic of TAT-CaM and cargo proteins with amino termini atleft. FIGS. 8B-8E show biolayer interferometry (BLI) analysis of TAT-CaMbinding to (FIG. 8B) purified endothelial nitric oxide synthase; (FIG.8C) CBS-β-Gal; (FIG. 8D) CBS-HRP; and (FIG. 8E) CBS-myoglobin. TAT-CaMwas biotinylated and bound to streptavidin (SA) sensors.Reference-subtracted raw data are rendered as points with fits to aglobal single-state association-then-dissociation model. Analyteconcentrations are noted for each trace. Association and dissociationphases were 300s in length. FIG. 8F shows dissociation data of theconstructs of FIGS. 8C-8E after dissociation in buffer only.

FIGS. 9A-9C show confocal imaging of cell penetration, where the cargois β-galactosidase (FIG. 9A), HRP (FIG. 9B), and myoglobin (FIG. 9C).

FIGS. 10A and 10B show cell penetration assay for SAP-CaM (FIG. 10A) andSAP(E)-CaM (FIG. 10B) using fluorescently labelled CBS-myoglobin ascargo.

FIGS. 11A-11F show biolayer interferomtery (BLI) analysis of sensorgramsof TAT-CaM 2.0 (FIG. 11A); TAT-calmodulin like protein 3 (FIG. 11B);SAP-CaM (FIG. 11C); SAP(E)-CaM (FIG. 11D); and TAT-troponin (FIG. 11E).FIG. 11F shows the calcium-dependent binding for all constructs in FIGS.11A-11E.

FIGS. 12A and 12B show transport of a cargo, CBS-tubulin, into livemyotubes in the presence of TAT-CaM (FIG. 12B) or absence of TAT-CaM(FIG. 12A).

FIGS. 13A and 13B show cell penetration assay for Cas9-CBS in thepresence of TAT-CaM (FIG. 13B) or absence of TAT-CaM (FIG. 13A).

FIG. 14 shows subcellular localization of a cargo comprising a nuclearlocalization sequence, according to one embodiment.

FIG. 15 shows one embodiment of a scheme of a Cas construct for nucleardelivery.

FIGS. 16A and 16B show BLI sensorgram ofTAT-CaM/CBS-tamavidin/biotinylated cargo complex assembly (FIG. 16A) anddelivery of CBS-tamavidin into BHK cells (FIG. 16B).

FIGS. 17A-17C show delivery of HPV E2 into SiHa cells (FIG. 17A),induction of senescence in SiHa cells (FIG. 17B), and reduction ofmetabolic activity (FIG. 17C).

FIG. 18 shows the structure of a targeted expression control construct,according to one embodiment.

The disclosed CPP-adapter constructs, schematically represented as

-   -   CPP ------ adapter -- adapter binding molecule ------ cargo        where the longer darker line indicates a covalent bond, and        where the shorter lighter line indicates a non-covalent bond,        overcomes these and other problems.

As only one non-limiting example, the construct of CPP=HIVtransactivator of transcription (TAT), the adapter=calmodulin (CaM), andcargo contains a calmodulin binding site, the construct overcomes thelimitations of covalently fused CPP-cargo constructs. Automatic internalrelease from the CPP is effected upon construct entry into a cell,because most mammalian cells maintain low resting calciumconcentrations, facilitating escape of the construct from endosomaldegradation.

The mechanism(s) by which CPP cellular uptake occurs is underinvestigation; however, several pathways appear to be used. In part,this reflects differences among the CPP themselves, but the same CPP maybe taken up by different pathways under different circumstances.

The initial interaction between cellular membranes and CPP-proteinconstructs is through interactions with membrane surface hydrophobiccomponents and/or negatively charged groups, such as phospholipids andheparin sulfate proteoglycans. The constructs that aremembrane-associated, but not yet translocated, are difficult todistinguish from translocated groups, except by advanced threedimensional methods (e.g., confocal microscopy). This difficulty has ledto artifacts in the study of CPP mechanisms. Once the CPP construct isassociated with the membrane surface, several translocation mechanismsmay be used. For example, there is evidence for clathrin dependentendocytosis, caveolin dependent endocytosis, and macropinocytosis fordifferent construct combinations, i.e., CPP and cargo.

Since discovery of the Tat peptide (TaTp), a variety of CPP have beenfound to enable transport of macromolecular cargos to both cells inculture and in animals. Well characterized CPP originating from the N-or C-termini of viral proteins include TATp, oligoarginines, MPGpeptides, Pep1, and VP22.

The TAT CPP derived from the carboxy terminus of the dopaminetransporter can translocate large cargos. For example, the 1,024 aminoacid E. coli β-galactosidase can be transported. It exists as a 464-kDahomotetramer with each subunit having five domains: jelly-roll typebarrel, two fibronectin type III-type barrels, a β-sandwich domain, anda TIM-type barrel domain that contains the catalytic site. The CPP tagcan enable translocation of such a multimer of modular components.

Versatile translocation systems that use CPP tags to produce novelsystems to transport cargo and manipulate the interior of cells aredisclosed.

Adapter molecules that make strong protein-protein interactions and arelinked to CPP are disclosed.

The inventive constructs provide a convenient and powerful method toperturb cell interiors. The constructs may contain many potential cargosand may be selective CPP. The invention is thus not limiting to aparticular component of the construct.

Non-limiting examples of the adapter, which is covalently bound to CPP,are disclosed. In one embodiment, the adapter is calmodulin or a relatedcalcium binding protein. In one preferred embodiment, the CPP is TAT oranother CPP listed in Table 2. In one embodiment, the adapter releasesits cargo after targeting to an interior cellular compartment. In oneembodiment, such targeting takes advantage of the low calciumenvironments in many compartments. In one embodiment, the adapter usesprotein-ligand interactions to bind the cargo by an adapter bindingmolecule, where the adapter binding molecule is non-covalently linked tothe adapter and is covalently linked to the cargo. In one embodiment,the construct of a CPP covalently bound to an adapter, which may bereferred to as a CPP tagged adapter, incorporates G protein subunitswith slow GTPase activity to release cargos internally after GTPhydrolysis. In one embodiment, the CPP tagged adapter molecule usesinternal enzymatic activity, e.g., kinase or phosphatase activity, torelease cargoes in the cell interior.

One embodiment is a method using CPP tagged adapters as previouslydescribed to deliver a cargo to the interior of cells either in cultureor in vivo. Applications include, but are not limited to, internalmeasurements of conditions including pH, calcium, oxygen, and nitricoxide concentration, and delivery of reagents to internal compartmentsto map the location of cell components using protein-proteininteractions. In one embodiment, the cargo delivered to internalcompartments detects and measures the presence of proteins and nucleicacids in vivo or in situ in cultured or isolated cells. In oneembodiment, the cargo is a fluorescence tagged Fab antibody fragment,and the target may be an internal pathogen so the method is used fordiagnosis of a pathology. In one embodiment, the cargo delivered tointernal compartments perturbs the cellular state, e.g., metabolicstate, developmental state, etc., in vivo or in situ. In one embodiment,the cargo is a constitutively active protein that is a phosphorylationmutant of kinases at activating sites. In one embodiment, the cargodelivered inside a cell and/or to internal compartments modifiesexpression, e.g., repressors or enhancers of gene expression, and smallRNAs such as siRNA and miRNA. In one embodiment, the cargo is deliveredto treat diseases resulting in part from internal pathogens. In oneembodiment, the cargo is an antibody or antibody fragment. In oneembodiment, the cargo is a specific enzyme, a ligand, or a small RNA. Inone embodiment, the cargo is delivered to treat a metabolic disorder orsymptoms of a metabolic disorder, such as delivering fah to treathereditary tyrosinemia. In one embodiment, the cargo is a constitutivelyactive modified kinase. In one embodiment, the cargo is delivered tomodify the genome of an organism.

Methods that integrate protein purification and cell entry use theattachment mechanism as an affinity ligand for chromatography.

In one embodiment where the cargo is a protein, the cargo has acalmodulin binding peptide as an N- or C-terminal extension, and is thenpurified on a calmodulin affinity column and delivered internally by CPPtagged calmodulin. In one embodiment where the cargo is a nucleic acidor small molecule, the calmodulin binding peptide is covalently linkedto the nucleic acid or small molecule.

In one embodiment, the cargo is a Cas protein linked to a calmodulinbinding peptide, and may also be linked to a nuclear localizationsequence, and CPP allows delivery of Cas to the cell interior. Cas maybe used for genome modification, including gene knock-out and geneknock-in modifications.

As one example, the method delivers a CRISPR /Cas complex to the cellinterior, using a Cas construct with a CaM binding peptide/adapterbinding molecule with TAT-CaM or other CPP-adapter. This example may beused for extensive downstream applications including, but not limitedto, protocols for using CRISPR/CRISPRi in living organisms, such asanimals and plants, cell lines with low efficiency of success usingconventional CRISPR-based techniques, antiviral applications,antiparasitic applications, chemotherapeutic agents using CRISPR geneediting to introduce mutations or CRISPRi to manipulate expression ofessential genes.

Other uses include delivery of therapeutics for autoimmune diseases withgenetic components (e.g. celiac disease, rheumatoid arthritis, etc.),potentially-fatal or disabling childhood genetic diseases (e.g.Tay-Sachs, PKU, Duchenne's muscular dystrophy, beta-thalassemia,sickle-cell anemia, etc.), and neurological disorders includingHuntington's disease, Alzheimer's disease, some forms of Parkinson'sdisease, amyotrophic lateral sclerosis, etc. CRISPR and CRIPSR1 is alsoused for topical therapies, e.g., for severe eczema and other skinconditions.

The method and construct may be used in industrial applications.Examples include rapid modification of single-celled organisms, e.g.bacteria and algae, biofuel production, rapid modification of organismsused in industrial chemical production, rapid modification of geneticorganisms used to produce biologics, e.g., insulin, growth hormone, etc.The compositions and methods also are used in agricultural applications,e.g., rapid genetic modification of livestock, optimization of animalhusbandry applications involving recombinant DNA, modification ofdomesticated crops for feedstocks and human food consumption,modification of crops used in biofuel applications, etc.

The CPP tagged adapters reversibly bind an adapter binding moleculetagged cargo by non-covalent bonds then release the cargo in the cellinterior, which may include targeting to one or more internal cellularcompartment, e.g., nucleus, mitochondria, peroxisomes, endoplasmicreticulum, etc. The stable but reversible adapter coupling the CPP to acargo has applications and advantages in safety, utility, and in ease ofcargo purification. The term ‘adapter’ has been used in reference to theCPP itself, not to a coupling intermediate.

Ideal adapters are small, stable, readily purified proteins capable ofstrong interaction with the cargo, either alone by intrinsicprotein-protein interactions, or via an adapter binding molecule, e.g.,a group such as biotin covalently bound to the cargo. Thisadvantageously allows a cargo protein to be purified by affinitychromatography using an N- or C-terminal extension, and the sameextension can be used to mediate cargo binding to the CPP taggedadapter.

This also provides other advantages. This scheme allows production ofcargos with only a single tag, rather than a CPP adapter and an affinitytag. This scheme also requires only a few CPP tagged adapters to bedeveloped to deliver many different cargos; significant because thedirect CPP tagged versions of many potential cargos carry a potentialrisk to workers involved in their purification due to the cell membranepermeability enhancement. Production of a limited number of relativelybenign CPP-adapter constructs, under well-controlled conditions,provides a significant safety factor, and the adapter-cargo complex needonly be assembled at the point of use, and in cases where constructformation is faster than uptake by cells, even being added separately tocell cultures.

The CPP-adapter-cargo construct can be designed to dissociate oncellular internalization. One convenient way of doing this is to use anadapter that responds to the internal cellular conditions. Othermethods, e.g., an unstable linkage, autocatalytic dissociation,photodissociation, etc., are also possible. The use of calcium bymammalian cells as a signal provides an avenue for cargo release; cellinteriors are normally maintained at very low levels of calcium byATP-driven pumps, and cells contain a variety of calcium biosensors thatrespond to transient calcium increases to tightly bind and releasetarget peptides. In one embodiment, the adapter protein is a calciumbiosensor such as calmodulin.

Calmodulin (CaM) is a small (16.7 kDa), soluble, heat resistant,multifunctional calcium biosensor protein that is the major mediator ofcalcium signaling in mammalian cells. When calcium is present, the CaMprotein folds into a dumbbell-shaped configuration with two connectedglobular regions; each end of the globular “dumbbell” contains twocalcium-binding EF hands. The alpha helix connector breaks, and closesaround targets containing a 17 amino acid canonical motif or one ofseveral alternative target motifs. CaM binds to targets with highaffinity, in the picomolar range, and is typically diffusion limited.CaM is the archetypical member of the EF hand-calmodulin superfamily ofcalcium signaling proteins. It is easy to produce CaM site-directedmutants and constructs; production of novel CaM constructs providesunique and valuable reagents.

The invention extends the usefulness of TAT peptide constructs, andrelated CPP constructs, by expressing TAT fusions of small adapters thatstrongly bind other molecules. Specifically, TAT-calmodulin is readilytaken up in whole organisms and by cells in culture, such as CHO cells.TAT was used as the initial CPP tag because of prior success inproducing TAT tagged proteins that are taken up by mammalian cells.Other CPP tagged calmodulins, such as antennapedia-CaM, SAP-CaM andSAP(E)-CaM, an engineered anionic version of SAP (Martin et al.,Chembiochem, 12(6), 896-903 (2011)), as well as TAT fusions with otherCaM binding proteins, such as TAT-troponin and TAT-calmodulin-likeprotein 3 (CALM3) have been made and shown to work in nearly identicalfashion.

FIG. 1 shows a scheme for the uptake of cargo, e.g., payload, covalentlytagged with a cell penetrating peptide (CPP) by cells. The inventivesystem and method uses adapters that provide strong protein-proteininteractions as a convenient and powerful method to perturb cellinteriors with a broad palette of selectively membrane permeable probes(FIG. 2). Common and inexpensively produced adapters are modified byintroducing a CPP tag, enabling any protein that binds the adapter to bemoved into cells. It is relatively easy and safe to express and purifyproteins with a tag that binds to a coupling protein with high affinity.Some tags allow rapid purification of the protein chosen for deliveryusing a one-step affinity column.

Delivery of proteins to the interior of cells has numerous applications.In addition to mapping the location of the components of living cellswith fluorescent tags, the availability of a system capable oftranslocating proteins into the cell interior enables detection ofinternal components in real time in living cells, and provides tools formanipulation of signaling pathways and gene expression by allowing theintroduction of constitutively active kinases, repressors, andenhancers. Viruses may be intracellularly detected and destroyed. Themetabolic state and/or expression profiles of cells may be altered,resulting in wide medical application.

Green Fluorescent Protein (GFP) and other fluorescent proteins may betagged. GFP and engineered variants and homologs, are powerful tools forcellular interior labeling; they are readily purified. GFP is typicallyexpressed after transfection with the appropriate vector, but many celltypes are resistant to transfection. In one embodiment, the cargodelivered is a fluorescent probe such as a GFP fusion containing a sitethat recognizes an internal target and a tag recognized by a CPPadapter, e.g., a calmodulin binding peptide recognized by TAT-CaM. Suchprobes are widely used, in part because they can be expressed inmammalian cells after transfection with a shuttle vector, andspontaneously generate a fluorophore inside the cells. The ability todeliver external probes broadens the possibilities.

Many protein types can be labeled with commercially available customfluorophores, e.g., the Alexa series, and introduced into the cellinterior compartments with CPP tags. Tagged proteins may be followed inthe cell with confocal microscopy. More demanding investigationsincluding FRET and fluorescence lifetime experiments may be performed.As FIGS. 3A and 3B show, in FRET experiments, components are labeledwith fluorophores chosen so that the emission spectrum of one, the donorD, is heavily overlapped with the excitation spectrum of the other, theacceptor A. If the labeled molecules associate in the cell, Forsterenergy transfer will cause the acceptor to fluoresce when the donor isexcited by pumping its absorbance lines, as FIG. 3A shows. If D and Aare not in proximity, there is no emission from A, as FIG. 3B shows.This provides information about complex formation in cells. FRETexperiments can be performed inside cells using two different GFPvariants, but it would be advantageous to use CPP adapters to deliver apair of proteins labeled with different synthetic fluorophores. Pairedfluorophores optimized for FRET, such as Alexa and DyLight, have farbetter properties, e.g., yield and spectral overlap, than the engineeredGFP variants. An important advantage is that they are small andintroduce much less steric interference than a GFP fusion.

In lifetime experiments, a fluorophore is repeatedly excited by a pulsefrom a laser and the fluorescence decays are collected, yielding thelifetimes of the fluorophore in all environments. Typically three orfour environments can be readily distinguished with lifetimes in the 50ps to 5 ns range and contributions as low as a few percent.

Calmodulin is remarkable for its high sequence conservation; only fourother proteins are more conserved in eukaryotes. Mammalian calmodulinsare identical, and C. elegans calmodulin is 96% identical to its humanhomolog. Sequence homology conservation is primarily driven by retentionof target specificity, not by the requirement for calcium binding andassociated organization into the characteristic dumbbell shape. Becausecalmodulin binds to many Ca²⁺ activated targets in cells, the ability ofthe targets and calmodulin to co-evolve is severely restricted.

FIGS. 4A and 4B show structures of calcium-calmodulin bound to acanonical target peptide (FIG. 4A) and in the dumbbell-shapedconformation in the absence of target (FIG. 4B). The central helixbreaks during recognition and binding, allowing calmodulin to wraparound the target. The protein is less ordered in the absence of calcium(not shown).

As the FIG. 5 alignment shows, sequence similarity within thecalmodulin-EF hand superfamily is much lower; identity within the fourhuman sequences shown is about 20%. The sequence variation within thesuperfamily allows members to recognize and regulate distinct targets inresponse to a single ionic signal. It permits using the differentspecificity of superfamily members to produce EF hand based adaptersthat are specific to different target sequences; all these targets areabout 17 AA in length because of the dimensions of the folded EF handproteins, but the amino acid sequences of the targets are different.This is important because it confers potential to address differentcargos to different cellular compartments. FIG. 6 shows the amino acidsequence of TAT-CaM, a CPP tagged calmodulin. The short CPP bindingsequence (SEQ ID NO: 26) is located directly before the NOT1 site, whichis followed by CaM (SEQ ID NO: 21).

Target proteins are delivered to the interior of cells with CPP labeledcalmodulin. Initial demonstrations were designed to use neuronal nitricoxide synthase (nNOS) and CaM kinase; both enzymes are activated bycalcium/calmodulin, and both can be purified on a calmodulin column. CaMkinase isoforms have monomer molecular masses of about 41 kDa; thetruncated CaM kinase II (New England Biolabs) has a molecular mass of 36kDa. However, CaM kinases form very large quartenary complexes of 400kDa-600 kDa, making them an exacting test for the calmodulin mediatedtranslocation system, comparable to beta-galactosidase. The nNOS activedimer has a molecular mass of about 322 kDa. Both nNOS and CaM kinaseproteins can be readily labeled with high quantum yield fluorophoresthat have distinctive spectral signatures, allowing evaluation of theiruptake and cellular distribution. These target proteins were selectedbecause they contain a calmodulin binding motif, but most proteins canbe produced with a small calmodulin binding tag at the N or C terminuswithout significantly affecting their activity, or like nNOS with aninternal tag associated with an exposed surface loop.

FIGS. 7A and 7B show confocal microscopy images demonstrating uptake ofa fluorescence labeled enzyme, neuronal nitric oxide synthase (nNOS),mediated by a CPP linked calmodulin adapter. Projection confocal imageof labeled nNOS three hours after onset of TAT-CaM mediated uptake byBHO cells. The indicated nucleus is stained blue; labeled nNOS isstained yellow with DyLight 540. FIG. 7A shows nNOS added without CPPadapter. Background still shows stained nNOS after washing with media.Some nNOS adheres to the cell surface; three-dimensional cross sectionsshow no nNOS inside cells. FIG. 7B shows that in the presence ofTAT-CaM, a large amount of nNOS is rapidly and actively pumped insidethe cell, clearing the intracellular space and protecting nNOS fromremoval by washing. The cell boundary is now visible because cytoplasmis stained by released nNOS. three dimensional cross sections confirmthat labeled nNOS is inside the cells. Circles (yellow) inside cells arelabeled endosomes.

There was rapid uptake of a novel cargo in which a calmodulin bindingsite is attached to cargos of myoglobin, horseradish peroxidase, andb-galactosidase. All these cargos are rapidly taken up by mammaliancells, and all rapidly enter the cytoplasm. Calcium triggered releasesolves the endosomal entrapment problem because cargos are released fromtrapped CPP when the calcium concentration drops. This is evidenced bycargo escape from endosomes to the cytoplasm while TAT-CaM remainstrapped, as shown below in FIG. 9.

TAT tagged calmodulin was initially produced as purified His-taggedcalmodulin using His tag and nickel column. Calmodulin bindingsite-containing cargos may be purified using a calmodulin affinitycolumn, a 17 amino acid canonical sequence calmodulin binding sequencebound with high affinity in the presence of calcium. Calmodulin withoutthe His tag can be produced by affinity chromatography, binding to thecolumn in the presence of calcium, and eluting with the calciumionophore EDTA.

FIG. 8A shows design of TAT-CaM and cargo proteins, according to oneembodiment, schematic of TAT-CaM and cargo proteins with amino terminiat left. FIGS. 8B-8E show biolayer interferometry (BLI) analysis ofTAT-CaM binding to (FIG. 8B) purified endothelial nitric oxide synthase;(FIG. 8C) CBS-β-Gal; (FIG. 8D) CBS-HRP; and (FIG. 8E) CBS-myoglobin.Biolayer interferometry (BLI) was performed using a ForteBio (Menlo ParkCalif., USA) Octet QK using SA sensors. Assays were done in 96 wellplates at 25° C. Volumes of 200 μL were used in each well. Ligands wereloaded onto sensors for 300-900 s followed by baseline measurements inbinding buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol) for 300s. Association was measured by dipping sensors into solutions of analyteprotein and was followed by moving sensors to buffer only to monitordissociation. Binding was fit to a global 1:1association-then-dissociation model using GraphPad Prism 5.02. TAT-CaMwas biotinylated and bound to streptavidin (SA) sensors.Reference-subtracted raw data are rendered as points with fits to aglobal single-state association-then-dissociation model. Analyteconcentrations are noted for each trace. Association and dissociationphases were 300s in length. FIG. 8F shows that after dissociation inbuffer only, sensors were moved to buffer containing 10 mM EDTA. Therapid dissociation phases of the 1 μM samples for each cargo protein areshown. Binding is shown as I/O specific binding to reconcile the varyingmagnitudes of different analytes.

The prototype CPP-adapter, TAT-CaM (New Echota Biotechnology, GeorgiaUSA), is encoded by a pET19b-based vector containing a cleavableHis-tag, the cell penetrating sequence from the HIV transactivator oftranscription fused to calmodulin via a GGR linker. Calmodulin wasselected as the prototype because mammalian cells typically maintain lowresting cytoplasmic Ca²⁺ levels, allowing rapid release of cargo afterinternalization. TAT-CaM was expressed and purified from E. coliBL21(DE3)pLysS using metal affinity chromatography without detergents.Cargo proteins were expressed in BL21(DE3)pLysS from pCal-N-FLAG-basedplasmids (Agilent Technologies, CA, USA) and included myoglobin (Myo),horse radish peroxidase (HRP) and β-galactosidase (β-Gal), each with anamino-terminal vector-encoded calmodulin binding site (CBS). Proteinswere purified over a calmodulin sepharose column (GE Healthcare) andexchanged into binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 10%glycerol, 1mM CaCl₂). For cell penetration assays, cargo proteins werelabelled with DyLight 550 according to the manufacturer's protocol.Unreacted label was removed via a dye binding column.

TAT-CaM bound to endothelial nitric oxide synthase via the native CaMbinding site, with affinity similar to wild-type CaM as assayed withbiolayer interferometry (BLI), an optical biosensing technique similarto surface plasmon resonance (FIG. 8B). All cargos bound CaM with lownanomolar affinity (FIG. 8C-E). TAT-CaM and cargo proteins dissociatedrapidly upon exposure to EDTA (k_(off)˜0.1 s⁻¹, FIG. 8F), i.e. theTAT-CaM-CBS interaction is functioning indistinguishably from wild typeCaM. All analytes exhibited negligible binding to sensors withoutTAT-CaM. Rate and affinity constants determined from single-state globalfits are listed in Table S1.

TABLE S1 Kinetic parameters for sensorgrams shown in FIG. 8. K_(D) (nM)k_(on) (M⁻¹ s⁻¹) k_(off) (s⁻¹) k_(off) (EDTA) (s⁻¹) eNOS 15 1.2 × 10⁴1.8 * 10⁻⁴ ND β-Gal 73 9.2 × 10³ 6.7 * 10⁻⁴ 0.05 HRP 160 1.7*10³ 2.8 *10⁻⁴ 0.2 Myo 0.9 5.9 × 10⁴ 5.5 * 10⁻⁵ 0.09 ND, not determined butpreviously reported to be ~0.11 s⁻¹.

For confocal studies, as shown in FIG. 9, 1 pM each of TAT-CaM andfluorescently-labelled cargo protein in buffer containing 1 mM CaCl₂were added to subconfluent BHK cells and incubated for 1 hour, afterwhich cells were washed three times in phosphate buffered saline with 1mM CaCl₂. This produced very high loading suitable for imaging;enzymatic activity requires much smaller levels of Tat-CaM and cargo.Treated cells were washed three times in phosphate buffer whichcontained 1 mM CaCl₂. Cells were labeled with NucBlue (LifeTechnologies) to visualize the nuclei. Cargo uptake into cell interiorswas assayed using an inverted Zeiss LSM700 confocal microscope equippedwith a 40× Neofluar-Plan objective (NA=1.3). Pinholes for eachfluorophore were set at 1.0 Airy Units (29 microns), and SP 490 and LP615 filters were used to acquire the NucBlue (Blue channel) and DyLight550 (Red Channel) signals, respectively. Z-stacks of both Tat-CaMtreated and untreated cells were acquired, and later analyzed forincorporation of fluorescently labeled cargo into the cytoplasm.Orthogonal projections of Z-stacks were then generated using Zeiss ZENsoftware, which allowed for viewing both treated and untreated cellsalike at the same depth within the cell, relative to the diameter of thenucleus. Using the diameter of the nucleus as a landmark, the Z-planechosen for analysis corresponded to approximately the mid-point depth ofthe nucleus. Finally, the DyLight 550 signal was analyzed separately(FIG. 9, left panel, white signal), and merged with NucBlue (FIG. 9,right panel, red (DyLight 550) and white (NucBlue), respectively).

FIGS. 9A-C show confocal imaging of cell penetration, where the cargo isβ-galactosidase (FIG. 9A), HRP (FIG. 9B), and myoglobin (FIG. 9C). BHKcells were treated for 1 hour with the indicated DyLight 550fluorescently labeled cargo proteins (white in left panel, red channelin right panel), in either the absence or presence of TAT-CaM, washedand imaged live. Orthogonal projections were generated and the centerimages presented are optical sections set at a similar depth of thenucleus (NucBlue staining, white, right panel), as determined byposition within the Z-stack. Comparison of TAT-CaM-treated vs. untreatedcells indicates that cargo proteins are entering into the cell, and arelocalized primarily to the cytoplasm. Scale bars in all panels=20 μm.

As shown in FIG. 9, all cargo proteins were delivered to the interiorsof the cells and showed significant cytoplasmic distribution, indicatingefficient penetration and escape from endosomes. The fluorescentlylabelled cargo proteins without TAT-CaM showed some adherence to thesurfaces of cells, but no discernable penetration at the samecytoplasmic depth as that observed cells treated with TAT-CaM (FIG.9A-C).

The alternative of CPP directly attached to a cargo, and previous workusing CPP attached to cargoes by covalent or, in a few cases,non-specific non-covalent interactions, have several drawbacks. Onedrawback is additional handing of potentially toxic CPP. Anotherdrawback is CPP remaining on the tag after internalization, causing thecargo to remain associated with the input machinery and preventingendosomal escape. Previous methods require toxic, laborious, andirreversible covalent crosslinking or assembly of irreversiblehydrophobic complexes.

In one embodiment, the cargo is tagged with an adapter recognized moiety(e.g., a calmodulin binding peptide) using standard cross linkingmethods. This embodiment is an alternative to integrated affinitypurification and CPP-adapter attachment for proteins that are producedin-house. For example, any commercial proteins, even lacking a CaMbinding site, can readily be tagged and rendered cell permeable bycovalent crosslinking.

Cell penetrating agents mediate penetration of the plasma membrane,allowing delivery of macromolecular cargos to cell interiors. However,until this invention, cell penetrating agents such as CPP have lagged indevelopment for each of research, diagnostic, and therapeuticapplications, hindered by their poor cargo delivery and lack of release.With the inventive system and method, there is efficient intracellulardelivery and endosomal escape of user-defined cargos, including but notlimited to protein cargos. For example, three different cargo proteinswere successfully delivered to the cytoplasm of BHK cells, demonstratingfeasibility of numerous applications in living cells includingalteration of signaling pathways and gene expression.

While there are over 25 CPP clinical trials underway, including one inPhase III, CPP have largely disappointed for reasons includingnon-penetration, limited endosomal escape, and requirements forhydrophobic cargos. The disclosed invention is a novel technology thatsolves or ameliorates all of these problems using a novel CPP-adapterprotein fusion. TAT-Calmodulin (TAT-CaM) is used to non-covalently bind,deliver and release cargo into the cytoplasm. The strategy is generallyapplicable to any soluble protein and also be used to delivernon-protein cargos. Assays can be performed in real time with live cellswithout significant cytotoxicity. The invention greatly expands theapplications of CPPs.

As described above, CPP other than TAT can also be used. FIG. 10 showsresults of a cell penetration assay for SAP-CaM (FIG. 10A) andSAP(E)-CaM (FIG. 10B) using fluorescently labelled CBS-myoglobin ascargo. BHK cells were treated for one hour with DyLight 550fluorescently labeled cargo protein CBS-myoglobin. The images areoptical sections set at a similar depth of the nucleus. DyLight550-tagged CBS-myoglobin is shown (left panel), nuclei are stained withNucBlue (center panel), and the cytoplasm is marked with CellTrackerGreen CMFDA dye (right panel). Orthogonal projections are shown to theright and top of each panel, demonstrating that the cargo penetrated thecell and was distributed throughout the cytoplasm, as opposed to adheredto the outside of the cell; see ‘+SAP-CaM’ (FIG. 10A) and ‘+SAP(E)-CaM’(FIG. 10B). Control experiments without CPP-adapter are labelled‘−SAP-CaM’ (FIG. 10A) and ‘−SAP(E)-CaM’ (FIG. 10B), and show nosignificant penetration, as expected.

FIG. 11 shows biolayer interferomtery (BLI) analysis of sensorgrams ofadditional described constructs. FIG. 11A shows TAT-CaM 2.0 (a modifiedversion of TAT-CaM of FIG. 6, where the Not1 site, and the GGR itencoded, are removed), FIG. 11B shows TAT-calmodulin like protein 3,FIG. 11C shows SAP-CaM, FIG. 11D shows SAP(E)-CaM, and FIG. 11E showsTAT-troponin. Ligands were biotinylated and tethered to streptavidinsensors. All analytes were CBS-myoglobin except in FIG. 11E usingtroponin inhibitory peptide-myoglobin. Analyte concentrations rangedfrom 1 μM to 63 nM, as indicated. Fits are shown to a one-stateassociation-then-dissociation model. Kinetic and affinity constants forCPP-adapter binding to CBS-myoglobin (or TIP-myoglobin) are shown in thetable below.

TAT- TAT- SAP- SAP(E)- TAT- Constant CaM 2.0 CALL3 CaM CaM Troponink_(on) (M⁻¹s⁻¹) 4900 6100 8300 9400 3700 k_(off) (s⁻¹) ND 1.8 × 10⁻⁴ 1.7× 10⁻⁴ 1.3 × 10⁻⁴ ND K_(D) (nM) ND 29.8 20.6 13.3 ND k_(off) (EDTA) 0.170.07 0.11 0.20 0.018 (^(s−)1)

FIG. 11F shows the calcium-dependent binding for all constructs in FIGS.11A-E, where the sensor was exposed to the highest concentration ofanalyte in FIGS. 11A-E and was dipped into buffer containing EDTA toremove calcium. Rapid dissociation was observed for each complex (koffabout 10⁻¹ s⁻¹ for all except TAT-troponin, which was 1.8×10⁻² s⁻¹).

The inventive complexes can be used to introduce a cargo into cells thatare difficult to transfect, such as primary cultures. As shown in FIG.12, the inventive constructs can be used to deliver cargo into myotubes,where myotubes were treated for one hour with DyLight 550 fluorescentlylabeled α-tubulin-CBS, in either the absence (FIG. 12A) or presence(FIG. 12B) of TAT-CaM, washed, and imaged live. The center panels showoptical sections set at a similar depth of the nucleus (NucBlue stainingshown by arrow in center panel), as determined by position within theZ-stack. Cytoplasmic compartments in live cells were visualized usingCellTracker Green CMFDA dye (shown by arrow in right panel). Comparisonof TAT-CaM-treated (FIG. 12B) with untreated cells (FIG. 12A) indicatesthat the cargo protein entered into the cell and localized primarily tothe cytoplasm.

In one embodiment, the cargo delivered by the CPP tagged adapter is amodulator, either activator or repressor, of transcription. In oneembodiment, the cargo is a probe that measures a property of the cellinterior, e.g., an oxidation monitor, NO sensors, pH sensor. In anotherembodiment, the cargo is a kinase, phosphatase or other enzyme, whichmay be modified to be constitutively active. In another embodiment, thecargo itself delivers another cargo such as tamavidin, caspase, etc.

Other cargos, including liposomes and their contents, nucleic acids,inhibitors, and drugs can also be delivered by extensions of the samemethods, e.g. using DNA binding proteins with calmodulin binding N or Cterminal extensions. In one embodiment, the cargo is a nucleic aciddelivered using a DNA or RNA binding protein with an adapter bindingmolecule. In another embodiment, the cargo is a drug or other smallmolecule delivered using a protein or other scaffold that binds thesmall molecule and is equipped with an adapter binding molecule.

Secreted peptide hormones such as insulin, bradykinin, EGF, etc. form alarge category of endocrine signal molecules controlling diverse andnumerous cellular processes. Many of these signal pathways areautoregulated by a negative feedback process that preferentiallyinternalizes hormone receptors with bound ligands.

The inventors were first to recognize and use cell signaling moleculesas CPP as cell penetrants. Peptide endocrine and paracrine signals arecell penetrating agents (CPA) if they promote receptor internalization.The prototype is insulin.

Compositions including a CPA-adapter construct combination, such asinsulin-calmodulin, are also disclosed. This is a distinct embodiment ofthe invention, in contrast to CPP-adapter technology using establishedCPPs, e.g., TAT-calmodulin or TAT-CaM. The embodiments described asusing CPPs can also be constructed using CPAs.

In some embodiments, signal molecule-based CPA may not be as effectiveas existing CPP for general internalization of molecules, but theyprovide the advantage of specifically targeting the vesicles in whichinternalized receptors are stored prior to recycling or degradation.This permits specific labeling experiments, FRET experiments, andmanipulation of the specific internal compartment to modify the fate ofinternalized receptors.

Peptide hormones are not unique as endocrine and paracrine signals, andreceptors for both larger molecules, e.g., mediating low densitylipoprotein uptake or ephrin signaling, and small molecules may be used.Epinephrine in an example of a small molecule endocrine signal thatinduces receptor internalization. The β-2-adrenergic receptor alsoserves as a well-established example of G protein coupled receptorinternalization in response to a small molecular ligand. Other examplesof ligand induced internalization include the folate receptor, theN-methyl-D-aspartic acid (NMDA) receptor, opiate receptors such as MOPr,and the CB1 cannabinoid receptor. All of these ligands are coupled toother proteins, such as the adapter CaM, and used to drive the uptake ofcargos into the specific endosomes that contain the ubiquinatedreceptors. This permits study of trafficking, and also potentiallyaffect the fate of receptors, which can either be recycled or shipped tolysosomes for degradation.

In addition to receptors, transporters are often regulated by ligandactivated internalization. Examples include autoregulation of dopamineuptake. This process is initiated by external ligands including cocaine,amphetamines, and melittin. Antidepressants induce internalization ofthe serotonin transporter in serotonergic neurons. The norepinephrinetransporter is also regulated by ligand induced internalization.Specialized cell penetrants may be comparable to the previouslydisclosed adapters. Other transporters, e.g., the endothelial glucosetransporters, are internalized and recycled from the cell interior inresponse to internal signals, e.g., endothelial glucose transport isregulated by internal glucose. This may be used for, e.g., neuroscienceinvestigations.

The CRISPR/Cas gene editing system is an important advance in researchand potential therapeutics. A major obstacle for both is that deliveryof CRISPR reagents, e.g. Cas9 and guide RNA, is usuallytransfection-based, which is difficult or impossible in many situations.The inventive method delivers the components of the CRISPR system,rescuing the function and allowing genome editing in recalcitrantsystems.

The clustered regularly interspaced short palindromic repeats,abbreviated CRISPR, are short DNA sequences characterized by repeatedpalindromic sequences. Palindromic repeats are separated by short DNAspacers, which may have been acquired from viral or plasmid DNA. TheCRISPR/Cas system allows prokaryotes to degrade exogenous DNA fromplasmids and phages. CRISPR/Cas has been developed as an inexpensive,powerful and transformative gene editing technique due to its wideapplicability as a both a tool and potential therapeutic. For geneediting, guide RNA and Cas9 protein must be expressed or delivered tothe cell interior.

Current use of the CRISPR/Cas system is severely limited by thefeasibility of delivering essential components to the cell interior.Delivery is currently by transfection or viral transduction ofeukaryotic cells with genes coding for Cas9 and for guide RNA; whileeffective for some applications, it is unsuccessful in others, eitherbecause of inefficient transfection/transduction or poor geneexpression. In contrast, as shown in FIG. 13, Cas9 delivery is achievedusing the described constructs. Specifically, FIG. 13B shows that, usingCas9-CBS and TAT-CaM, Cas9 was effectively delivered inside BHK cells,where BHK cells were treated for one hour with 1 pM DyLight 550-labelledCas9. FIG. 13A shows the control without TAT-CaM.

CPP- or CPA-adapters deliver components to the cytoplasm and target themto specific cellular compartments, including the nucleus. For example,as FIG. 14 shows, a cargo comprising CBS-myoglobin with a consensusnuclear localization sequence from SV40 Large T-antigen (SEQ ID NO: 1),complexed with TAT-CaM, was efficiently transport to the nucleus.Specifically, 1 μM DyLight 550-labelled CBS-myoglobin with a consensusnuclear localization signal was complexed with 1 μM TAT-CaM anddelivered to BHK cells. The cytoplasm is stained with CytoTracker Green,the nucleus with NucBlue and the NLS-CBS-myoglobin in red. The pinkcolor, indicated by the arrow, demonstrated co-localization ofDyLight-labelled CBS-NLS-myoglobin and the NucBlue nuclear stain, andnuclear localization. The right panel of FIG. 14 shows the orthogonalprojection, demonstrating nuclear localization.

This nuclear localization allows the extension of CRISPR/Cas technologyto recalcitrant systems. The application of CPP-adapters andCPA-adapters required production of novel Cas protein constructs. Ingeneral, such constructs include a Cas protein, an adapter binding site,and a nuclear targeting signal sequence. In one embodiment, theconstruct contains an N terminal nuclear localization sequence (NLS), acalmodulin binding sequence, and a Cas encoding sequence separated byshort spacers. In some cases it may be necessary to also deliver thedouble stranded RNA component, either with a directly bound adapterrecognition tag or using an RNA binding protein.

Examples of common nuclear localization sequences include PKKKRKV SEQ IDNO: 1 and KR[PAATKKAGQA]KKKK SEQ ID NO: 2, a bipartite NLS where the KRand KKKK are the determinant residues. A representative example of acanonical calmodulin binding sequence is the nNOS KRRAIGFKKAEAVFSAKLMSEQ ID NO: 3 sequence (Bredt and Snyder, Proc. Natl. Acad. Sci. 87(2)682-685 1990). Similar sequences 17-20 residues long are high affinityligands for calcium replete calmodulin; other CaM binding motifs, e.g.,the IQ motif, are also possible. FIG. 15 shows one embodiment of aschematic of a Cas construct for nuclear delivery. This construct can bedelivered by the disclosed TAT-CaM CPP adapter system using a constructof the HIV derived CPP TAT and calmodulin.

Outgrowths of CRISPR-Cas include CRISPRi and CRISPR based expressioncontrol. In CRISPRi, an inactive Cas is targeted to specific genomic DNAsequences by guide RNA. This technology can be extended by deliveringthe components to the nucleus using the inventive CPP-adaptertechnology. This will interfere with expression of a specific gene orgenes by preventing transcription through steric inhibition, e.g., ofpromoter binding. CRISPRi is thus analogous to RNAi suppression ofexpression, but at the level of transcription rather than translation,useful to target integrated retroviral sequences into the host genomefor transcriptionally silencing viral DNA sequences.

In one embodiment, the invention is a composition comprising an inactiveCas analog of the construct shown in FIG. 15, and a method forexpression suppression.

Expression controls using Cas-enhancer and Cas-repressor constructs areclosely related to CRISPRi. These are targeted by guide RNA to locationsadjacent to the binding sites for promoters or enhancers, greatlyincreasing the effective affinity of these agents for their bindingsites on chromosomal DNA. Unlike CRISPRi, downregulation is independentof steric effects by Cas binding and in some cases would be moreeffective.

It may be desirable to improve delivery of CRISPR guide RNAs to thenucleus. Three potential methods for achieving this are to deliver apreformed Cas-RNA complex, deliver an alternative RNA binding proteincomplexed with guide RNA, or deliver the guide RNA using apeptide-nucleic acid (PNA) adapter binding moiety bonded to the guideRNA, itself, or to the complement of the guide RNA, which can annealwith a sequence in the RNA. Release of guide RNA from thedouble-stranded cargo inside the cell can be done by matching theannealing temperature to the experimental conditions. A more effectivescheme would lower the temperature of the cells to favor annealing, thenreturn cells to 37° C. to dissociate the cargo RNA from the adapter.

As previously described, cellular internalization is stimulated withligands and is used to translocate cargo into the cell interior. Not allreceptors and transporters are internalized, except on a long time scaleas part of general protein turnover. For example, many somatostatinreceptor types are not internalized because of ligand binding but theirinternalization can be forced with CPP attached to receptor ligands by acrosslinker. In one embodiment, a CPP is linked by a crosslinker to aCPA. The structure of the crosslinker may be an amino acid chain 5-20residues in length, depending on the size of the membrane proteins.Alternatively, materials other than peptide, e.g., carbohydrates, couldbe employed as a crosslinker. One end of the crosslinker would becovalently attached to a CPP, and one end would be attached to atransporter or receptor ligand or a CPA. An adapter, such as CaM, can beintegrated into the linker to allow detachment inside the cell. This isschematically shown as follows:

-   -   CPP-crosslinker-transporter    -   CPP-crosslinker-receptor ligand    -   CPP-crosslinker-cell penetrating agent

Many of the most important techniques available in research andtherapeutics are hindered by inability to get nucleic acids across cellmembranes. The roster includes transfection of cells with DNA,inhibition of translation, hence expression, by RNAi, CRISPR-Casediting, and Cas mediated control of expression. Nucleic acid targetvariants using the inventive system and method solve many of theseproblems.

Many DNA and RNA binding proteins are available that recognize single ordouble stranded nucleic acids. A calmodulin binding site (CBS)-nucleicbinding protein is a potential vehicle for DNA or RNA import. Thedrawback is release; reliance on equilibrium either reduces the importefficiency, i.e., low affinity, or slows the release rate, i.e., highaffinity. Additional affinity considerations are subsequently disclosed.Some prokaryotic nucleic acid binding proteins are calcium sensitive.The visinin-like protein, important in neurons, is an EF hand proteinthat binds double stranded RNA, and is a neuron-specificcalcium-dependent double-stranded RNA-binding protein. High affinity CaMbinding transcription activators provide an alternative strategy; CaMTAsare DNA binding proteins that are calcium activated via Ca⁺² release, solow internal calcium would release a CaMTA-nucleic acid complex fromTAT-CaM, triggering immediate nucleic acid release from CaMTA.

Biotin, a water-soluble B vitamin, is used as a labelling molecule,particularly for end-labeling of synthetic nucleic acids. Avidin, aprotein with high affinity for biotin, e.g. streptavidin, is used asbiotin-binding proteins in laboratory processes. CPP-adapters can beused in tandem with calmodulin-binding site (CBS)-avidin fusions to bindand deliver biotinylated nucleic acids. CBS-tamavidin has been producedand delivered to BHK cells, as shown in FIG. 16. Specifically, FIG. 16Ashows biolayer interferometry sensorgram ofTAT-CaM/CBS-tamavidin/biotinylated cargo complex assembly, where phase 1is TAT-CaM binding to sensors; phase 2 is CBS-tamavidin binding toTAT-CaM; and phase 3 is biotinylated cargo protein binding toCBS-tamavidin. The traces vary only in the concentration of biotinylatedcargo protein: 2 μM, 1 μM, and 500 nM, and all data are referencesubtracted. CBS-tamavidin did not bind sensors in the absence of TAT-CaMand biotinylated cargo did not bind TAT-CaM-saturated sensors. As FIG.16B shows, TAT-CaM delivered CBS-tamavidin into BHK cells (CBS-tamavidinand nuclei shown by arrows). Other embodiments include monomericstreptavidins. CBS-avidins can also be used to bind and deliver proteinsthat are not expressible or active as CBS-fusions but can bebiotinylated, e.g. antibodies.

A construct CPP-nucleic acid or CPA-nucleic acid can be made usingchemical crosslinking. Such a construct is a CPP or CPA adapter systemanalogous to TAT-CaM if the nucleic acid is complementary to a sequencein the cargo polynucleotide, allowing duplex formation. An example is aCPP-DNA construct such as TAT-TTTTTTTTTTTT (TAT-SEQ ID NO: 4). Such aconstruct would bind tightly to the polyA tail of messenger RNA attemperatures below the melting point of the duplex. This can be amechanism for facilitated endosomal escape, loading at slightly reducedtemperatures (25° C.-32° C.) followed by return to 37° C., where theduplex will dissociate. For polyA-polyT interactions, 10-15 bases arelikely to be optimal.

TAT-TTTTTTTTTTT (TAT-SEQ ID NO: 4)     |||||||||||    AAAAAAAAAAAAAA-cargo (SEQ ID NO: 5-cargo)Other nucleic acids can be internalized using custom TAT-DNA constructsthat anneal to target sequences in the cargo DNA. Alternatively, forapplications that tolerate a short additional sequence, e.g., a polyAtail or a shorter CG rich tail, a generic TAT-DNA construct could beused. While TAT-DNA is easier to work with and much less sensitive todegradation, TAT-RNA and TAT morpholinos are also useful.

For cell culture experiments, high affinity between CPP and their targetand/or receptor is optimal as long as internal release is sufficientlyfast to internalize cargo into internal compartments. This is likelyalso true for experiments targeting small volumes in large animals, orin experiments with small animals, e.g., C. elegans. For experimentstargeting larger volumes, lower affinity CPP are superior because theyare not be taken up by the first few layers of cells they encounter. Ingeneral, targeting larger volumes of tissue require large amounts ofCPP-adapter-cargo complex and CPP with lower affinity. CPP withprogressively altered amino acid sequences to obtain altered affinityand kinetics can be evaluated. TAT-CaM delivers cargo to the cytoplasmwithin about five minutes, and may be in endosomes in about a minute orless. A low affinity mutant of TAT, or another CPP, allows diffusion andcirculation to reach a much larger cell population. Constructs similarto TAT CaM may be designed that would act on the time scale of hours.

Conversely, the affinity of the CPP-adapter for the cargo constructshould be high to optimize import efficiency; this is very effective forcargos with a built in release, e.g., Ca²⁺ sensitivity, pH sensitivity,temperature sensitivity, autocleavage, etc. For cargo without such amechanism, a lower affinity would permit a compromise between efficientimport into endosomes and release from the membrane. This can be readilyaccomplished by modifying the linkage between adapter and cargo.

The inventive system and method has use in anti-cancer strategies,including those subsequently described and also those includingtelomerase suppression, suppression of motility factors that promotemetastasis, and suppression or enhancement of oncogene products asappropriate.

The enzyme telomerase extends chromosomal DNA by adding repeats of thesequence TTAGGG as telomeres to the 3′ end of chromosomes, non-codingextensions that are progressively removed during cell divisions becauseof the inability of DNA polymerase to add bases onto the 3′ end of DNA.Mammalian telomerases are only active during meiosis, and the length ofthe telomeric extension determines how many times cells can dividebefore the coding region is degraded and the cells senesce.

In one embodiment, an active telomerase is delivered to the nucleus,greatly extending the lifetime of cells. Unlike cancer cells, thesecells would not be immortalized, because telomerase would not beexpressed and the level of activity would fall with time. Cell culturescould be retreated, however, with each treatment extending their life.This is a great advance in cell and tissue culture. In addition, stemcell lifetimes could be greatly extended, not only delaying senescencebut producing an essentially unlimited population of cells from a smallinitial sample. This is of great value for researchers, and allows newtherapeutic approaches in diverse areas, e.g., wound healing toanti-agathics.

The inventive system and method enhances existing chemotherapeutictreatment regimens and protocols for the treatment of malignantcarcinomas. Many carcinomas result from increased or decreased activityof bHLH transcription factors such as Twist, a developmentally criticaltranscription factor that, when abnormally regulated, can induceepithelial cells to change from a senescent, sessile state to amalignant motile state. The nuclear transcription co-factors Akirin-1and/or Akirin-2 is/are a regulator of transcription factor activity in avariety of contexts. Targeting and reducing Akirin-2 levels usingnucleic acid-based techniques have been demonstrated to increasesensitivity of Twist-regulated cancer cells in vivo. However, theadministration and control of stoichiometry of nucleic acid-basedmolecules is problematic using conventional technologies.

Using the inventive CPP-based technology overcomes the myriad issueswith delivery of nuclei acids to carcinoma cells in vivo. For example,generating a construct consisting of the transcription factor (TF)interacting domain (ID) of Akirin-1 and/or Akirin-2, fused with anuclear localization signal, cell-penetrating peptide, and CaM bindingsite (AkirinID-CPP). Using the disclosed inventive delivery system, thefusion protein is introduced to tumor cells including, but not limitedto, glioblastoma, neuroblastoma, retinoblastoma tumor cells, and thefusion protein is delivered to the cell and subsequently translocated tothe cell nucleus. Once inside the cell, this interaction domainout-competes endogenous TF/Akirin interaction and favors TF/AkirinID-CPPinteractions instead, which weakens the tumor cells and renders themmore susceptible to parallel treatments. This method is utilized for allpotentially mechanistically meaningful domains of Akirin-1 and Akirin-2,once they are identified.

The inventive system and method delivers senescence inducing proteins invirally derived cancers and other anti-viral therapeutics, also withresearch applications.

Cervical carcinoma is the third most common cancer, representing about16% of all cancers among females. About 90% of cervical cancers arecaused by human papilloma virus (HPV), most frequently (75%) by twostrains: HPV type 16 and 18. E6 and E7 are the major viral oncogenes. E6protein binds tumor suppressor p53, targeting it for ubiquitin-mediateddegradation. E6 also stimulates telomerase activity. E7 protein bindsp105^(Rb) and other retinoblastoma tumor suppressor proteins. Thecombined effect of the activities of these proteins leads to aggressiveproliferation.

Early viral transcription produces E2 protein, which repressesexpression of E6 and E7. However, during integration into the hostchromosome leading to oncogenesis, the gene encoding it is disrupted,which leads to increases in E6 and E7 expression and proliferation.Differential activities of E6 and E7 in HeLa cells were previouslycharacterized and induced replicative senescence by superinfection witha recombinant SV40 virus encoding bovine E2.

The disclosed inventive senescence inducing cargo protein CBS-E2, orcalmodulin binding site fused to HPV protein E2, is used in tandem withTat-CaM. For example, as FIG. 17 shows, TAT-CaM delivered HPV E2 inducedsenescence in SiHa cells. FIG. 17A shows delivered fluorescentlylabelled CBS-E2 (indicated by arrow; magenta) aligning on the mitoticplate of a dividing cell. Cytoplasm is marked with CytoTracker Green andnuclei are labeled with NucBlue. FIG. 17B shows control with TAT-CaMonly (left panel), and TAT-CaM/CBS-E2 (right panel), whereTAT-CaM/CBS-E2 induced morphological changes indicative of senescence at72 hr. FIG. 17C shows metabolic activity reduction varies by theconcentration of E2, TAT-CaM only controls are insignificantly differentfrom no addition control (Ctrl), and 2 μM and 4 μM E2 causedsignificantly lower metabolic activity of the affected cells. CPP/E2offers many advantages over recombinant virus in terms of safety,stability and effective dosing. Applications include, but are notlimited to the following:

Elimination of HeLa contamination of cultured cells. HeLa cells, theoldest and most commonly used human cell line, has undergone horizontalgene transfer from HPV18 at five different sites in its hypertriploidgenome. It is well adapted to growth in tissue culture plates and easilycontaminates other lines, often overgrowing them. Users are often notaware of the overgrowth, leading to devastating but unknown impact on amultitude of projects; one report was 29% of 360 knowncross-contaminated cell lines were HeLa contaminated; another report wasabout 10-20% of all in vitro cell lines are contaminated with HeLa,resulting in numerous artifactual results across a wide array ofresearch efforts. CPP-E2 arrests the proliferation of HeLa in acontaminated cell line while allowing proliferation of the desiredcells, eliminating the contamination.

Tissue culture media additive. The stability of CPP-E2 makes it anexcellent additive for tissue culture media to prevent HeLacontamination. Its specificity for HPV-infected cells means that it isnon-toxic and non-interfering to other types of cells. There is a largeand growing global cell culture supplies market.

Topical antiviral. CPP-E2 is soluble, stable and non-toxic. It can beused as an antiviral additive to condom lubricants, diaphragm jellies,contraceptive sponges, representing a significant value-addedproposition to the global contraceptives market. CPP-E2 may also be usedas an additive to cleaning products.

Anti-cancer therapeutic. Papillomavirus infects keratinocytes, causingskin lesions. Given CPP-EP2 cell-penetrating capabilities, applicationsare delivery to tumors in vivo for cervical, skin, and other mucosal orcutaneous cancers.

Oncogenesis research tool. Delivery of E6 and E7 can induceimmortalization when, paired with induction of senescence, a suite oftools may be produced for study of cellular processes and eventssurrounding the immortalization/replicative senescence boundary.

Other applications include other antivirals including ones targetedagainst herpes, HIV, Epstein-Barr, hepatitis B, and hepatitis C.

Targeted expression control. The ability to load constructs directlyinto cell interiors makes it possible to deploy synthetic cargos notfound in nature and which cannot be expressed by transfected cells.Examples of these constructs can be used to up or down regulateexpression.

FIG. 18 schematically shows the structure of one embodiment of atargeted expression control construct. The CaM binding site can dockwith a construct such as TAT-CaM, importing it into the cell. The Nterminal nuclear localization signal targets the construct to thenucleus. In the nucleus, the attached complementary DNA sequence, whichcan be covalently attached but need not be as long as the attachment ishigh affinity, binds to a chromosomal sequence 3′ to the gene andassociated control sequences. This targets the repressor or enhancer tothe appropriate binding site, up or down regulating the target gene. Inprinciple, this is similar to CRISPRi but is simplified because only asingle molecule is needed rather than inactive mutant Cas and a guideRNA.

Many other applications exist for expression control. Exemplarynon-limiting examples include reduction of inflammatory proteins,repression of telomerase in cancer treatment, enhanced production ofenzymes where function has been compromised by low expression, etc.

Internalization of antifreeze proteins (AFP) and ice nucleation proteinsin organisms living in environments where the ambient temperature isbelow the freezing point of water; such organisms have evolvedantifreeze proteins to prevent cellular damage secondary to ice crystalformation. Antifreeze proteins interact with water molecules to preventfreezing by regulating the formation and shape of ice crystals. AFP maybe localized to the extracellular fluid, the cell surface, orintracellularly. Treatment with extracellularly-administered AFPs canimprove cryopreservation of embryos and a wide range of cell types.However, treatment with Type 1 AFP has led to increased destruction ofsome cell types and tissues during freezing, apparently due to formationof needle-like intracellular ice crystals.

Use of the inventive CPP-based technology intracellularly delivers awide range of AFP into cells destined for cryopreservation, withouttheir genetic modification. An example strategy fuses a calmodulinbinding site to an AFP, producing CBS-AFP to be used in tandem withTAT-CAM or other CPP-CAM construct. CBS-AFP has the potential to enhanceviability of cryopreserved cells and embryos by regulating intracellularice formation, and may increase the potential for cryopreservation ofwhole tissues and organs.

The class of ice nucleation proteins (INP) promote formation of icecrystals. CPP-based delivery of intracellular INP would render cellsmore susceptible to cold injury. In an analogous example strategy toCBS-AFP, CBS-INP is constructed by fusing a calmodulin binding site toan ice nucleation protein, and delivered intracellularly using TAT-CAM.CBS-INP could be a useful adjuvant to cryosurgery.

Oct4, Soc2, Klf4 and c-Myc, the Yamanaka Factors are highly expressed inembryonic stem cells. Their forced expression in differentiated cellscan return those cells to a stem cell state, creating inducedpluripotent stem cells (iPSC). Previous approaches used retroviralvectors to deliver cDNAs expressing these reprogramming factors. Whileeffective in the laboratory, this gene delivery strategy createspotential problems, particularly if iPSC are to be used in a therapeuticcontext. The inventive cargo system and method bypasses this issue bydelivering proteins directly to the cell. This enormous benefit isextended to the direct reprogramming of differentiated cells into othercell types, e.g., neurons, islet cells, etc.

Other CPP-cargo strategies rely on covalent linkage or nonspecifichydrophobic linkers that are susceptible to getting trapped by membraneassociation as the CPP itself may not be released into the cytoplasm.Even non-hydrophobic CPP are likely to be tightly bound to membraneproteins associated with the import machinery, which would be expectedto greatly hinder endosomal escape by covalently attached cargoproteins. One group estimates that a fraction of 1% of TAT-fused cargosescape endosomes. High-affinity but reversible noncovalent attachment ofcargos overcomes trapping effects via Ca²⁺-dependent dissociation,allowing rapid and efficient cargo distribution to the cytoplasm eventhough the CPP may remain attached to the membrane.

Cytotoxicity is also a major drawback with current CPP, particularlygiven the concentrations necessary to attain observable endosomalescape. With cytotoxic effects of TAT becoming significant in the tensof pM, the fact that TAT-CaM effects significant cytoplasmicdistribution of cargos at 1 μM without an increase in cell death asmeasured by Trypan Blue exclusion (data not shown) is particularlyexciting. Release of cargo is the rate limiting step in endosomalescape.

The time frame of cargo delivery using CPP-adapters is an hour or less.This contrasts to the time required to transfect cells; CPP-adapteraugmentation is roughly two orders of magnitude faster thantransfection, making possible time course experiments that could neverbe previously attempted.

An wide array of applications can be developed from the inventive methodand system. CPP-adapters can allow for subcellular addressing, e.g.delivery of a transcription factor to the nucleus, with kinetics,dosing, toxicity and other parameters as well delivery of a wide arrayof cargos.

The following references are expressly incorporated by reference hereinin their entirety:

Fonseca et al. Recent advances in the use of cell-penetrating peptidesfor medical and biological applications. Adv. Drug Deliv. Rev. 61 (2009)953-964.

Johnson et al. Therapeutic applications of cell-penetrating peptides.Methods Mol. Biol. 683 (2011) 535-551.

Sebbage. Cell-penetrating peptides and their therapeutic applications.Biosci. Horizons 2 (2009) 64-72.

Gautam et al. CPPsite: a curated database of cell penetrating peptides.Database (Oxford) 2012, bas015 (2012).

Green and Loewenstein. Autonomous functional domains of chemicallysynthesized human immunodeficiency virus Tat trans-activator protein.Cell 55 (1988) 1179-1188.

Trabulo et al. Cell-penetrating peptides-Mechanisms of cellular uptakeand generation of delivery systems. Pharmaceuticals 3 (2010) 961-993.

Wadia et al. Transducible TAT-HA fusogenic peptide enhances escape ofTAT-fusion proteins after lipid raft macropinocytosis. Nat Med 10 (2004)310-315.

Mitchell et al. Polyarginine enters cells more efficiently than otherpolycationic homopolymers. J. Pept. Res. 56 (2000) 318-325.

Morris et al. A new peptide vector for efficient delivery ofoligonucleotides into mammalian cells. Nucleic Acids Res. 25 (1997)2730-2736.

Chaloin et al. Design of carrier peptide-oligonucleotide conjugates withrapid membrane translocation and nuclear localization properties.Biochem. Biophys. Res. Commun. 243 (1998) 601-608.

Elliott and O'Hare. Intercellular trafficking and protein delivery by aherpesvirus structural protein. Cell 88 (1997) 223-233.

Montrose et al., Xentry, a new class of cell-penetrating peptideuniquely equipped for delivery of drugs Scientific Reports 3, Articlenumber: 1661 (2013)

Rickhag et al. Membrane-permeable C-terminal Dopamine TransporterPeptides Attenuate Amphetamine-evoked Dopamine Release J. Biol. Chem.288 (2013) 27534-27544.

Magzoub et al. Interaction and structure induction of cell-penetratingpeptides in the presence of phospholipid vesicles. Biochim. Biophys.Acta 1512 (2001) 77-89 S.

El-Andaloussi et al. Cell-penetrating peptides: mechanism andapplications, Curr. Pharma. Design 11 (2005) 3597-3611.

Wagstaff et al. Protein Transduction: Cell Penetrating Peptides andTheir Therapeutic Applications; Current Medicinal Chemistry, 13 (2006)1371-1387.

Weigel et al. PNAS Plus: Quantifying the dynamic interactions between aclathrin-coated pit and cargo molecules PNAS 110 (2013) E4591-E4600

Säälik et al. Protein cargo delivery properties of cell-penetratingpeptides. A comparative study. Bioconjug Chem.15 (2004)1246-53.

Krebs and Heizmann Calcium-binding proteins and the EF-hand principle,pp. 51-93 in Calcium A Matter of Life or Death. Krebs and Michalak Eds.,41(2007) Elsevier.

Stratton et al. Structural studies on the regulation of Ca2+/calmodulindependent protein kinase II. Curr Opin Struct Biol. 23 (2013) 292-301

Houdusse and Cohen. Target recognition by the calmodulin superfamily:Implications from light chain binding to the regulatory domain ofscallop myosin Proc. Natl. Acad. Sci. USA 92 (1995) 10644-47.

Usui et al. Vascular effects of novel calmodulin-related proteins thatmediate development of hypertension Folia Pharmacologica Japonica 141(2013) 85-89.

Geller et al. Molecular cloning and expression of inducible nitric oxidesynthase from human hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 90 (1993)3491-5.

Hudmon and Schulmand (2002). Neuronal Ca2+/Calmodulin-Dependent ProteinKinase II: The Role of Structure and Autoregulation in CellularFunction. Annual Review of Biochemistry 71 (2002) 473-510.

Nowak and Baylies. Akirin: a context-dependent link betweentranscription and chromatin remodeling. Bioarchitecture, 2 (2012)209-213.

Krossa et al. (2015). Down regulation of Akirin-2 increaseschemosensitivity in human glioblastomas more efficiently than Twist-1.Oncotarget. 6 (2015) 21029-45.

zur Hausen. Immortalization of human cells and their malignantconversion by high risk human papillomavirus genotypes. Semin. CancerBiol. 9 (1999) 405-11.

Mantovani and Banks. The human papillomavirus E6 protein and itscontribution to malignant progression. Oncogene 20 (2001)7874-7887.

Macville et al. Comprehensive and definitive molecular cytogeneticcharacterization of HeLa cells by spectral karyotyping. Cancer Research59 (1999) 141-50.

Capes-Davis et al. Check your cultures! A list of cross-contaminated ormisidentified cell lines. Int. J. Cancer 127 (2010) 1-8.

DeFilippis et al. Endogenous Human Papillomavirus E6 and E7 ProteinsDifferentially Regulate Proliferation, Senescence, and Apoptosis in HeLaCervical Carcinoma Cells. J. Virology 77 (2003) 1551-1563.

Lonn and Dowdy. Cationic PTD/CPP-mediated macromolecular delivery:charging into the cell Expert Opin. Drug Deliv. 12 (2015) 1627-36.

Glogau et al. Results of a randomized, double-blind, placebo-controlledstudy to evaluate the efficacy and safety of a botulinum toxin type Atopical gel for the treatment of moderate-to-severe lateral canthallines. Journal of Drugs in Dermatology, 11 (2012) 38-45.

Palm-Apergi et al., Do cell-penetrating peptides actually” penetrate”cellular membranes?. Molecular Therapy, 20 (2012) 695-97.

Lundberg et al., Cell surface adherence and endocytosis of proteintransduction domains. Molecular therapy, 8 (2003) 143-150.

Erazo-Oliveras et al., Improving the endosomal escape ofcell-penetrating peptides and their cargos: strategies and challenges.Pharmaceuticals, 5 (2012) 1177-1209.

Hirose et al. Transient focal membrane deformation induced byarginine-rich peptides leads to their direct penetration into cells.Molecular Therapy 20 (2012): 984-93.

McMurry et al. Weak interactions between Salmonella enterica FlhB andother flagellar export apparatus proteins govern type III secretiondynamics. PloS one 10 (2015): e0134884.

Abdiche et al. Determining kinetics and affinities of proteininteractions using a parallel real-time label-free biosensor. J. Anal.Biochem. 377 (2008) 209-217.

Sultana and Lee, Measuring Protein-Protein and Protein-Nucleic AcidInteractions by Biolayer Interferometry. Current Protocols in ProteinScience, 79 (2015) 19-25.

McMurry et al. Rate, affinity and calcium dependence of nitric oxidesynthase isoform binding to the primary physiological regulatorcalmodulin. FEBS Journal 278 (2011) 4943-54.

Cardozo et al. Cell-permeable peptides induce dose-and length-dependentcytotoxic effects. Biochimica et Biophysica Acta (BBA)-Biomembranes 1768(2007) 2222-34.

Mathisen et al. Visinin-like protein (VILIP) is a neuron-specificcalcium-dependent double-stranded RNA-binding protein. J. Biol. Chem.274 (1999) 31571-76.

Takahashi and Yamanaka. Induction of pluripotent stem cells from mouseembryonic and adult fibroblast cultures by defined factors. Cell 126(2006) 663-676.

Takahashi et al. Induction of pluripotent stem cells from fibroblastcultures, Nat. Protoc. 2 (2007) 3081-89.

Applicants incorporate by reference the material contained in theaccompanying computer readable Sequence Listing identified as077875.53_ST25.txt, having a file creation date of Jan. 10, 2017 9:23A.M. and file size of 14 kilobytes.

The embodiments shown and described in the specification are onlyspecific embodiments of inventors who are skilled in the art and are notlimiting in any way. Therefore, various changes, modifications, oralterations to those embodiments may be made without departing from thespirit of the invention in the scope of the following claims. Thereferences cited are expressly incorporated by reference herein in theirentirety.

1. A composition for intracellular delivery of a biomolecule, thecomposition comprising a cell penetrating peptide (CPP) or cellpenetrating agent (CPA) covalently linked to an adapter, and a cargomolecule covalently linked to an adapter binding molecule, thecomposition formed by non-covalent linkage between the adapter andadapter binding molecule.
 2. A composition comprising a cell penetratingpeptide (CPP) or cell penetrating agent (CPA) covalently linked to anadapter, the adapter non-covalently linked to an adapter bindingmolecule, a cargo molecule covalently linked to the adapter bindingmolecule, the composition for intracellular delivery of the cargo.
 3. Acomposition for intracellular delivery of a biomolecule, the compositioncomprising a cell penetrating peptide (CPP) or cell penetrating agent(CPA) covalently linked to an adapter, the adapter non-covalently linkedto an adapter binding molecule, the adapter binding molecule covalentlylinked to a cargo.
 4. The composition of claim 1 where the adapter iscalmodulin or a calcium binding protein and the adapter binding moleculeis a calmodulin binding peptide.
 5. The composition of claim 1 where theCPP is selected from the group consisting of Tat, penetratin,transportan, Dat, VP-22, amphipathic peptides, MPG, Pep-1, MAP, SAP,PPTG1, arginine rich peptides, oligoarginine, hCT (9-32), SynB, andPvec.
 6. The composition of claim 1 where the CPA is selected from areceptor or transporter ligand.
 7. The composition of claim 6 where thereceptor is selected from the group consisting of insulin receptor, beta2-adrenergic receptor, folate receptor, the N-methyl-D-aspartic acid(NMDA) receptor, opiate receptors, cannabinoid receptor, andcombinations thereof.
 8. The composition of claim 6 where thetransporter is selected from the group consisting of dopaminetransporter, serotonin transporter, norepinephrine transporter,endothelial glucose transporter, and combinations thereof.
 9. Thecomposition of claim 1 where the adapter binding molecule is furtherattached to a sequence that localizes the cargo to a cellular locationor organelle that is selected from the group consisting of nucleus,peroxisome, mitochondria, endoplasmic reticulum, Gogli, and combinationsthereof.
 10. The composition of claim 9 where the sequence is a nuclearlocalization sequence.
 11. The composition of claim 1 where the cargo isselected from the group consisting of a protein, a drug, a liposome, anucleic acid, and combinations thereof.
 12. The composition of claim 1where the non-covalent linkage between the adapter and the adapterbinding molecule is reversible.
 13. The composition of claim 1 where atleast one characteristic of an intracellular environment promotesreversal of the non-covalent linkage between the adapter and the adapterbinding molecule, resulting in release of the cargo from the CPP. 14.The composition of claim 13 where the characteristic is an intracellularcalcium concentration.
 15. The composition of claim 1 where the cargo isselected from the group consisting of a modulator of transcription inthe cell, a probe that measures a property of the cell interior, and anenzyme.
 16. The composition of claim 15 where the enzyme is a kinase ora phosphatase.
 17. The composition of claim 15 where the enzyme ismodified to be constitutively active.
 18. The composition of claim 15where the probe is an oxidation monitor, a nitric oxide (NO) sensor, ora pH sensor.
 19. The composition of claim 11 where the cargo is anucleic acid and the adapter binding molecule is covalently linked to anucleic acid binding protein.
 20. A method for delivering a cargo insidea cell, the method comprising forming a complex by contacting a cellpenetrating peptide (CPP) or cell penetrating agent (CPA) covalentlylinked to an adapter, with a cargo molecule covalently linked to anadapter binding molecule, under conditions suitable for forming anon-covalent bond between the adapter and adapter binding molecule, andcontacting the cell with the complex under conditions sufficient toresult in delivery of the cargo inside the cell.
 21. The method of claim20 where the cell penetrating peptide (CPP) or cell penetrating agent(CPA) covalently linked to an adapter and the cargo molecule covalentlylinked to an adapter binding molecule are added separately to culturemedia containing the cell, and the complex forms in the culture media.22. The method of claim 20 where the adapter is calmodulin or a calciumbinding protein and the adapter binding molecule is a calmodulin bindingpeptide.
 23. The method of claim 20 where the CPP is selected from thegroup consisting of Tat, Penetratin, Transportan, Dat, VP-22,Amphipathic peptides, MPG, Pep-1, MAP, SAP, PPTG1, arginine richpeptides, Oligoarginine, hCT (9-32), SynB, and Pvec.
 24. The method ofclaim 20 where the CPA is selected from the group consisting of areceptor or transporter ligand.
 25. The method of claim 24 where thereceptor is selected from the group consisting of insulin receptor, beta2-adrenergic receptor, folate receptor, the N-methyl-D-aspartic acid(NMDA) receptor, opiate receptors, and cannabinoid receptor.
 26. Themethod of claim 24 where the transporter is selected from the groupconsisting of dopamine transporter, serotonin transporter,norepinephrine transporter, and endothelial glucose transporter.
 27. Themethod of claim 20 where the adapter binding molecule is also bound to alocalization sequence.
 28. The method of claim 27 where the localizationsequence is a nuclear localization sequence.
 29. The method of claim 20where the cargo is at least one of a protein, a drug, a liposome, or anucleic acid.
 30. The method of claim 20 where the non-covalent linkagebetween the adapter and the adapter binding molecule is reversible. 31.The method of claim 30 where at least one characteristic of anintracellular environment promotes reversal of the non-covalent linkagebetween the adapter and the adapter binding molecule, resulting inrelease of the cargo from the CPP.
 32. The method of claim 31 where thecharacteristic is an intracellular calcium concentration.
 33. The methodof claim 20 where the cargo is selected from the group consisting of amodulator of transcription in the cell, a probe that measures a propertyof the cell interior, an enzyme, and combinations thereof.
 34. Themethod of claim 33 where the enzyme is a kinase or a phosphatase. 35.The method of claim 33 where the enzyme is modified to be constitutivelyactive.
 36. The method of claim 33 where the probe is selected from thegroup consisting of an oxidation monitor, a nitric oxide (NO) sensor, apH sensor, and combinations thereof.
 37. The method of claim 29 wherethe cargo is a nucleic acid and the adapter binding molecule iscovalently linked to a nucleic acid binding protein.